Enzyme assay methods are widely applied in various fields such as biochemistry, clinical chemistry, homeland security, molecular biology, pathology, and toxicology. The sensitivity of enzyme assays capable of sensing analyte existing in a small volume of a sample is dependent on various factors such as the components as well as concentration of buffer solution, incubation time, the reactivity between enzyme and substrate, temperature, and detection method (e.g., absorbance, chemiluminescence, fluorescence). It is possible to rapidly quantify trace levels of analytical materials when these factors are optimized.
The condition of each factor can be optimized independently, but these factors interact in a complementary manner to enhance the accuracy, precision, reproducibility, and sensitivity. For example, it is well-known that chemiluminescence detection is more sensitive than absorbance and fluorescence, which have relatively high background noise. An enzyme assay with chemiluminescence detection is faster than those with absorbance and fluorescence because the incubation time of the former is shorter than that of the latter. Thus, it is desirable to be able to optimize enzyme assay methods to more easily and rapidly detect low-level concentrations of analytes as well as to be able to quantify higher concentrations.